The present invention is related to novel nucleic acids comprising telomerase reverse transcriptase (TERT, or TRT) cis-acting transcriptional control sequences. The present invention is further directed to methods of using these cis-acting transcriptional control sequences, for example, to drive heterologous gene sequences; to modulate the level of transcription of TERT or to isolate novel trans-acting regulatory factors which bind to and modulate the activity of a TERT promoter.
The following discussion is intended to introduce the field of the present invention to the reader. The citation of various references in this section is not to be construed as an admission of prior invention.
It has long been recognized that complete replication of the ends of eukaryotic chromosomes requires specialized cell components (Watson (1972) Nature New Biol. 239:197; Olovnikov (1973) J. Theor. Biol. 41:181). Replication of a linear DNA strand by conventional DNA polymerases requires an RNA primer, and can proceed only 5xe2x80x2 to 3xe2x80x2. When the RNA primer bound at the extreme 5xe2x80x2 ends of eukaryotic chromosomal DNA strands is removed, a gap is introduced, leading to a progressive shortening of daughter strands with each round of replication. This shortening of telomeres, the protein-DNA structures physically located on the ends of chromosomes, is thought to account for the phenomenon of cellular senescence or aging of normal human somatic cells in vitro and in vivo (see, e.g., Goldstein (1990) Science 249:1129; Martin (1979) Lab. Invest. 23:86; Goldstein (1969) Proc. Natl. Acad. Sci. USA 64:155; Schneider (1976) Proc. Natl. Acad. Sci. USA, 73:3584; Harley (1990) Nature 345:458-460; Hastie (1990) Nature 346:866-868; Counter (1992) EMBO J. 11:1921-1929; Bodnar (1998) Science 279:349-52).
The length and integrity of telomeres is thus related to entry of a cell into a senescent stage (i.e., loss of proliferative capacity). Moreover, the ability of a cell to maintain (or increase) telomere length may allow a cell to escape senescence, i.e., to become immortal.
The maintenance of telomeres is a function of a specific DNA polymerase known as telomerase reverse transcriptase (TERT, or TRT). Telomerase is a ribonucleoprotein (RNP) that uses a portion of its RNA moiety as a template for telomere repeat DNA synthesis (see, e.g., Morin (1997) Eur. J. Cancer 33:750). Consistent with the relationship of telomeres and TERT to the proliferative capacity of a cell (i.e., the ability of the cell to divide indefinitely), telomerase activity is detected in immortal cell lines and an extraordinarily diverse set of tumor tissues, but is not detected (i.e., was absent or below the assay threshold) in normal somatic cell cultures or normal tissues adjacent to a tumor (see, U.S. Pat. Nos. 5,629,154; 5,489,508; 5,648,215; and 5,639,613; see also, Morin (1989) Cell 59:521; Shay (1997) Eur. J. Cancer 33:787; Kim (1994) Science 266:2011). Moreover, a correlation between the level of telomerase activity in a tumor and the likely clinical outcome of the patient has been reported (see e.g., U.S. Pat. No. 5,639,613; Langford (1997) Hum. Pathol. 28:416).
Telomerase activity has also been detected in human germ cells, proliferating stem or progenitor cells, and activated lymphocytes. In somatic stem or progenitor cells, and in activated lymphocytes, telomerase activity is typically either very low or only transiently expressed (see, e.g., Chiu (1996) Stem Cells 14:239; Bodnar (1996) Exp. Cell Res. 228:58; Taylor (1996) J. Invest. Dermatol. 106:759).
Accordingly, human TERT (hTERT, hTRT) is an ideal target for treating human diseases relating to cellular proliferation and senescence, such as cancer. The cis-acting transcriptional control elements of TERT provided herein also allow for the identification of trans-acting transcription control factors. Moreover, the discovery and characterization of the TERT cis-acting sequences provide opportunities to develop useful disease therapies. The present invention fulfills this and other needs.
The invention provides an isolated, synthetic, or recombinant polynucleotide comprising a human telomerase reverse transcriptase (hTERT) promoter sequence. In alternative embodiments, the promoter sequence comprises at least 15, 50, 100, 150, 200, 250, 500, 1000, 2500 or at least 13,000 bases as set forth in residues 44 to 13490 in SEQ ID NO:1 or SEQ ID NO:2. Other embodiments include sequences starting within about one to 5 nucleotides of a translation start codon and ending at about 50, 100, 150, 200, 250, 500, 1000, 2500 or 13500 nucleotides upstream of the translation start codon in SEQ ID NO:1 or SEQ ID NO:2. The promoter sequence can comprise the sequence as set forth in residues 44 to 13490 in SEQ ID NO: 1.
The invention provides an isolated, synthetic, or recombinant polynucleotide comprising a human telomerase reverse transcriptase (hTERT) promoter or a mouse telomerase reverse transcriptase (mTERT) sequence operably linked to a transcribable sequence. The transcribable sequence can encode a protein other than hTERT or mTERT. The protein can be a cellular toxin. In one embodiment, the protein has activity that is not itself toxic to a cell, but which renders the cell sensitive to an otherwise nontoxic drug; e.g., the protein can be a Herpes virus thymidine kinase. Alternatively, the transcribable sequence can encode a protein that is detectable by fluorescence, phosphorescence, or by virtue of its possessing an enzymatic activity. The detectable protein can be firefly luciferase, alpha-glucuronidase, alpha-galactosidase, chloramphenicol acetyl transferase, green fluorescent protein, enhanced green fluorescent protein, and the human secreted alkaline phosphatase.
The invention also provides a method for screening for a compound that binds to TERT promoter, such as an hTERT or an mTERT promoter, comprising the following steps: (i) providing an isolated, synthetic, or recombinant polynucleotide comprising a TERT promoter sequence and a test compound, (ii) contacting the polynucleotide with the test compound, and (iii) measuring the ability of the test compound to bind to the polynucleotide.
The invention also provides a method for a method for screening for a compound that modulates a TERT promoter, such as hTERT or mTERT promoter activity, comprising the following steps (i) providing a first polynucleotide comprising an isolated, synthetic, or recombinant TERT promoter sequence operably linked to a transcribable second nucleotide, and a test compound, (ii) contacting the polynucleotide with the test compound, and (iii) measuring the ability of the test compound to modulate transcription of the second nucleotide. The transcribable sequence can encode a protein. The protein can be detectable by fluorescence or phosphorescence or by virtue of its possessing an enzymatic activity. The detectable protein can be firefly luciferase, alpha-glucuronidase, alpha-galactosidase, chloramphenicol acetyl transferase, green fluorescent protein, enhanced green fluorescent protein, and the human secreted alkaline phosphatase.
The invention also provides a method for identifying a cis-acting transcriptional regulatory sequence that modulates a TERT promoter, such as hTERT or mTERT, promoter activity, comprising the following steps (i) providing a first construct comprising a first polynucleotide comprising an isolated, synthetic, or recombinant TERT promoter sequence operably linked to a transcribable second nucleotide, (ii) providing a second construct comprising a modification in a subsequence of the first polynucleotide of step 1 operably linked to a transcribable second nucleotide, and (iii) measuring independently under the same conditions the ability of the first construct and the second construct to induce transcription of the transcribable second nucleotide, wherein an increase or decrease in the ability of the second modified construct to induce transcription of the transcribable second nucleotide as compared to the first unmodified construct identifies in the second modified construct a subsequence acting functionally as a cis-acting transcriptional regulatory sequence that modulates TERT promoter activity.
The invention also provides a method for generating a cell that lacks a TERT promoter activity, comprising the following steps (i) providing a polynucleotide comprising an isolated, synthetic, or recombinant TERT promoter sequence; (ii) introducing into the cell the polynucleotide of step (i), and (iii) measuring ability of the cell to transcribe TERT message; wherein the inability of the cell to transcribe TERT message indicates that a cell that lacks TERT promoter activity has been generated.
The invention also provides a genetically engineered cell lacking TERT, e.g., hTERT or mTERT, promoter activity, produced by introducing into the cell a polynucleotide comprising an isolated, synthetic, or recombinant TERT promoter sequence.
The invention also provides a composition comprising an isolated nucleic acid molecule comprising a TERT promoter, wherein the promoter comprises about 100 to about 200, 200 to about 400, 400 to about 900, or 900 to about 2500, or 2500 to about 5000 nucleotides upstream of a transcriptional start site in SEQ ID NO:1 or SEQ ID NO:2.
The invention also provides a vector comprising a TERT promoter operably linked to a heterologous nucleic acid sequence, wherein the promoter comprises about 100 to about 200, 200 to about 400, 400 to about 900, or 900 to about 2500, or 2500 to about 5000 nucleotides upstream of a transcriptional start site in SEQ ID NO:1 or SEQ ID NO:2.
The invention provides a transformed cell comprising a TERT promoter operably linked to a heterologous nucleic acid sequence, wherein the promoter comprises about 100 to about 200, 200 to about 400, 400 to about 900, or 900 to about 2500, or 2500 to about 5000 nucleotides upstream of a transcriptional start site in SEQ ID NO:1 or SEQ ID NO:2. The heterologous nucleic acid can code for a cellular toxin. The cellular toxin can be a Herpes virus thymidine kinase, ricin, abrin, diphtheria, gelonin, Pseudomonas exotoxin A, tumor necrosis factor alpha (TNF-alpha), Crotalus durissus terrificus toxin, Crotalus adamenteus toxin, Naja naja toxin, and Naja mocambique toxin.
The invention also provides a method of expressing a heterologous nucleic acid sequence in a cell comprising: (a) transforming said cell with a vector or an expression cassette comprising a TERT promoter, wherein the promoter consists of about 100 to about 200, 200 to about 400, 400 to about 900, or 900 to about 2500, or 2500 to about 5000 nucleotides upstream of a transcriptional start site in SEQ ID NO:1 or SEQ ID NO:2, and wherein the promoter is operably linked to the heterologous nucleic acid sequence; and (b) growing said cell under conditions where the heterologous nucleic acid sequence is expressed in said cell.
The invention also provides an isolated nucleic acid molecule which hybridizes to SEQ ID NO:1 or SEQ ID NO:2 under stringent hybridization conditions. The nucleic acid can consist of about 100 to about 200, 200 to about 400, 400 to about 900, or 900 to about 2500, or 2500 to about 5000 nucleotides upstream of a transcriptional start site in SEQ ID NO:1 or SEQ ID NO:2. The stringent hybridization conditions can comprise a hybridization step comprising a salt concentration of about 0.02 molar at pH 7 and a temperature of about 60xc2x0 C. The stringent hybridization conditions can also comprise a washing step at 65xc2x0 C. in 0.1xc3x97SSC, 0.1% SDS. In an alternative embodiment, the stringent hybridization conditions are selected to be about 5-10xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
The invention also provides transgenic non-human animals comprising a TERT promoter sequence operably linked to transcribable sequence, such as a reporter gene. In a preferred embodiment, an hTERT promoter is operably linked to a reporter gene in a transgenic mouse.
A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification, the figures and claims.
All publications, GenBank deposited sequences, ATCC deposits, patents and patent applications cited herein are hereby expressly incorporated by reference for all purposes.